In many eukaryotic cells, the subcellular positioning and dynamics of membranous organelles is dependent on microtubules and microtubule- based transport. The arrangement of the microtubule cytoskeleton along the apico-basal axis of simple polarized epithelia suggests that these filaments provide a structural framework that both yields stability to themonolayer and provides a substrate for transport ofmaterials across the cell. It appears that transcellular movement in the basal-to-apical direction, i.e. toward microtubule minus ends, is highly dependent on microtubules, suggesting tht the motor protein, cytoplasmic dynein, andits activator, dynactin are key players in this process. Polarized epithelia are therefore a useful model system for in-depth study of dynein-and dynactin-based intracellular motility. Understanding of mechanisms ofmicrotubule dynamics and microtubule- based motility has been significantly extended by the application of video-enhanced microscopy techniques, as they allow evaulation in real time of complex subcellular behaviors. Studies of intracellular membrane traffic and endomembrane dynamics have benefited equally fromthis sort of analysis. Inthis proposal, a series of experiments designed to elucidate the transcytotic pathway in WIF-B cells and to determine how cytoplasmic dynein and dynactin contribute to transcellular movement in WIF-B and MDCK are proposed. Membrane dynamics will be visualized using a novel surface labeling technique that allow traffic from the basal surface to the apical surface to be observed directly in real time by video-enhanced fluorescence microscopy. A separate line of investigation will explore the mechanism of microtubule nucleationin WIF-B and MDCK cells. Many epithelia are post-mitotic and their microtubule cytoskeletons lack a centralfocus, suggesting that conventional centrosomal nucleating mechanisms may not be involved. Sites for microtubule nucleationwill be identified and it will be determined if their centrosomes have the capacity to nucleate and release microtubules in vitro and in vivo.